PROJECT SUMMARY/ABSTRACT The long-term goal of this project is to reveal the mechanisms by which complex odors are encoded, with a special focus on the initial stages of olfactory information processing. The research design takes advantage of the anatomical simplicity and powerful genetic toolkit of Drosophila melanogaster, which allows systematic molecular genetic analysis of olfactory circuits as well as in-depth physiological and behavioral analysis of olfactory function. The results may have major implications for the control of insect vectors of disease. The project focuses on ephaptic interactions, a novel, non-synaptic form of olfactory circuit communication, which take place between any two grouped olfactory receptor neurons (ORNs) housed in the same insect sensory hair (sensillum). Despite its ubiquity, how ephaptic communication regulates olfactory function and behavior is poorly understood. We recently provided the first description of the importance of ephaptic inhibition in insect olfaction. In the current study, we will first focus on defining the importance of ephaptic excitation. A systematic, functional survey will be performed to define the strength of ephaptic excitation between grouped ORNs (Aim 1). The respective electrotonic properties of grouped ORNs will also be determined (Aim 2). The strength of ephaptic interactions will be quantified between a pair of ORNs from both directions with a view to testing the hypothesis that ephaptic interactions are asymmetric across sensillum types. Furthermore, the ultrastructure of grouped ORNs will be described using serial block-face electron microscopy (SBEM) and 3D reconstruction imaging technologies (Aim 3). This Aim is designed to identify the biophysical factors that underlie asymmetric ephaptic interactions in a sensillum. Morphological features of an identified ORN, such as dendritic caliber, number of dendritic branches, as well as soma size, will be analyzed and compared between neighboring ORNs. The hypothesis that the physically larger ORN in a pair exerts stronger ephaptic interactions upon its neighbor will be tested. This Aim could also lead to critical technical breakthroughs to broaden the application of SBEM in illuminating the 3D ultrastructure of any identified cell in diverse tissues. Lastly, the functional importance of ephaptic interactions in odor-guided behavior will be determined (Aim 4). Specifically, we will extend our behavioral assay and define the role of ephaptic inhibition on courtship behavior in sensillum which houses ORNs responsive to pheromone cues. We will also perform the first test of the functional importance of ephaptic excitation between another ORN pair that mediates behavioral responses to food odors. The proposed research will determine the functional importance and biophysical principles of a novel form of olfactory circuit interaction mechanism. These findings have the potential to revolutionize our understanding of olfactory information processing in insects, and may reveal general principles that govern chemosensory behavior throughout the animal kingdom.